The present invention relates generally to the field of molecular biology, and in particular, skeletogenesis. More particularly, certain embodiments concern nucleic acid segments comprising a gene that encodes a novel osteoblast-specific transcription factor, designated Osf2/Cbfa1. In certain embodiments, the invention concerns the use of these polynucleotide and polypeptide compositions to regulate osteoblast differentiation, and stimulate bone tissue formation, growth, repair and regeneration. Methods are also provided for identifying Osf2/Cbfa1 and Osf2/Cbfa1-related genes and polypeptides from biological samples, as well as methods and kits for identifying compounds that interact with Osf2/Cbfa1 polypeptides or polynucleotides, as well as compounds that alter or inhibit osteogenesis in an organism.
1.2.1 Bone Development
Regulatory factors involved in bone repair are known to include systemic hormones, cytokines, growth factors, and other molecules that regulate growth and differentiation. Various osteoinductive agents have been purified and shown to be polypeptide growth-factor-like molecules. These stimulatory factors are referred to as bone morphogenetic or morphogenic proteins (BMPs), and have also been termed osteogenic bone inductive proteins or osteogenic proteins (OPs). Several BMP- (or OP-) encoding genes have now been cloned and characterized; these have been assigned the common designations of BMP-1 through BMP-8. Although the BMP terminology is widely used, it may prove to be the case that there is an OP counterpart term for every individual BMP (Alper, 1994). Likewise, additional genes encoding OPs and BMPs are still being identified.
BMPs 2-8 are generally thought to be osteogenic, although BMP-1 is a more generalized morphogen (Shimell et al., 1991). BMP-3 is also called osteogenin (Luyten et al., 1989) and BMP-7 is also called OP-1 (Ozkaynak et al., 1990). BMPs are related to, or part of, the transforming growth factor-xcex2 (TGF-xcex2) superfamily, and both TGF-xcex21 and TGF-xcex22 also regulate osteoblast function (Seitz et al., 1992). Several BMP (or OP) nucleotide sequences and polypeptides have been described in U.S. Pat. Nos. 4,795,804; 4,877,864; 4,968,590; and 5,108,753; including, specifically, BMP-1 disclosed in U.S. Pat. No. 5,108,922; BMP-2A (currently referred to as BMP-2) in U.S. Pat. Nos. 5,166,058 and 5,013,649; BMP-2B (currently referred to as BMP-4) disclosed in U.S. Pat. No. 5,013,649; BMP-3 in U.S. Pat. No. 5,116,738; BMP-5 in U.S. Pat. No. 5,106,748; BMP-6 in U.S. Pat. No. 5,187,076; BMP-7 in U.S. Pat. No. 5,108,753 and U.S. Pat. No. 5,141,905; and OP-1, COP-5 and COP-7 in U.S. Pat. No. 5,011,691 (each of which is specifically incorporated herein by reference in its entirety).
Other growth factors or hormones that have been reported to have the capacity to stimulate new bone formation include acidic fibroblast growth factor (Jingushi et al., 1990); estrogen (Boden et al., 1989); macrophage colony stimulating factor (Horowitz et al., 1989); and calcium regulatory agents such as parathyroid hormone (PTH) (Raisz and Kream, 1983). Skeletal development is a multi-step process. It includes patterning of skeletal elements, commitment of mesenchymal cells to chondrogenic and osteogenic lineages, followed by the terminal differentiation of precursor cells into three specialized cell types: the chondrocyte in cartilage, the osteoblast and osteoclast in bone. Many genes encoding either growth factors or transcription factors were shown through genetic studies in mice to control patterning of skeletal elements (Luo et al.; 1996a, 1996b). These genetic analyses showed also that mutations in these genes do not severely affect the differentiation of the skeleton specific cell types suggesting that patterning and cell differentiation in the skeleton are achieved through different genetic pathways. Consistent with this hypothesis, genes such as PTHrP and c-fos were shown to control chondrocyte and osteoclast differentiation respectively, without affecting skeletal patterning (Karaplis et al., 1994; Wang et al., 1992; Johnson et al., 1992). Little is known, however, about the molecular determinants specifically responsible for controlling osteogenesis, and in particular, osteoblast differentiation.
Analysis of Osf2, the osteoblast nuclear activity polypeptide that binds to OSE2, showed that it is immunologically related to the Cbfa transcription factors (Geoffroy et al., 1995; Merriman et al., 1995). The Cbfa proteins are the mouse homologues of Runt, a Drosophila pair-rule gene product required for neurogenesis and sexual differentiation (Gergen and Wieschaus, 1985; Kania et al., 1990). Runt and the Cbfa proteins have a high degree of homology in their DNA-binding domain, a 128-amino-acid long motif called the runt domain (Kagoshima et al., 1993). The mouse genome contains three known runt homologues encoding numerous isoforms with well-characterized expression patterns (Ogawa et al., 1993a; Bae et al., 1992; Wijmenga et al., 1995; Simeone et al., 1995). None of the described Cbfa transcripts has been shown to be expressed exclusively or predominantly in bone, suggesting that still unknown member(s) of the Cbfa family control osteoblast-specific expression of Osteocalcin. This prompted the search for such a novel member or members.
1.2.2 Transcription Factors
The control of all biological processes results from a balance between various positive and negative-acting factors which interact with DNA regulatory elements and with each other. These protein factors play a critical role in controlling the expression of proteins, and thus are critical to both normal and pathological processes. Understanding these protein factors and how they modulate gene expression is key to strategies for the development of agents to control disease initiation and progression.
Gene-specific transcription factors provide a promising class of targets for novel therapeutics directed to these and other human diseases for the following reasons. One, transcription factors offer substantial diversity. Over 300 gene-specific transcription factors have been described, and the human genome may encode as many as 3000. Hence, they provide as plentiful a target source as cell-surface receptors. Two, transcription factors offer substantial specificity. Each and every factor offers unique molecular surfaces to target. Three, transcription factors are known to be involved in human disease. For example, many tumors are associated with the activation of a specific oncogene. A third of known proto-oncogenes and three fourths of all anti-oncogenes are transcription factors.
Transcription factors are capable of sequence-specific interaction with a portion of a gene or gene regulatory region. The interaction may be directed sequence-specific binding where the transcription factor directly contacts the nucleic acid or indirect sequence-specific binding mediated or facilitated by other auxiliary proteins where the transcription factor is tethered to the nucleic acid by a direct nucleic acid binding protein. In addition, some transcription factor demonstrate induced or synergistic binding. A broad range of transcription factor-nucleic acid complexes provide useful targets. The gene and/or transcription factor may be derived from a host or from an infectious or parasitic organism. As examples, a host may be immunomodulated (e.g., by controlling inflammation or hypersensitivity) by modulating the DNA binding of a transcription factor involved in immune cell activation; or vital, bacterial, or other microbial disease progression may be inhibited by disrupting the DNA binding of a host, vital or other microbial transcription factor involved in vital or other microbial gene transcription.
What is lacking in the prior art, inter alia, are polynucleotide compositions that encode polypeptides that possess osteoblast-specific transcription factor activity. Also lacking are methods of regulating transcription of genes involved in skeletogenesis, and in particular, those involved in expression of osteoblast-specific genes.
The present invention overcomes these and other limitations in the prior art by providing for the first time, an osteoblast-specific transcription factor that regulates differentiation along osteoblastic lineages. The invention provides methods that overcomes the prior limitations that regulate transcription of genes involved in skeletogenesis.
In a first embodiment, the invention provides novel polynucleotide compositions comprising an Osf2/Cbfa1 gene, that encodes the first osteoblast-specific transcription factor identified, the Osf2/Cbfa1 polypeptide disclosed herein. Also provided are methods for the use of this gene and its regulatory sequences (including its promoter and enhancer elements) in the regulation and expression of specific genes, including those involved in osteogenesis. The invention also provides antibodies reactive with Osf2/Cbfa1, and a variety of related immunological methods, compositions, and devices. The methods of the invention also provide for the use of Osf2/Cbfa1 gene and Osf2/Cbfa1 polypeptide compositions in the regulation of heterologous genes positioned under the control of Osf2/Cbfa1 and Osf2/Cbfa-derived nucleic acid sequences. In illustrative embodiments, Osf2/Cbfa1 has been shown to regulate the expression of genes in mesenchymal cells that are required for osteoblast differentiation, and Osf2/Cbfa1 polypeptides have been shown to possess osteoblast-specific transcription factor activity.
In a second embodiment, the present invention provides a method of specifically transcribing a gene, and in particular, an osteoblast-specific gene. The method generally involves providing to a cell an amount of an Osf2/Cbfa1 composition effective to specifically transcribe the gene of interest. Such genes may be homologous or heterologous genes, and may include genes derived from a variety of sources, including mammalian sources. In exemplary embodiments, the inventors have demonstrated the occurrence of a variety of osteoblast-specific genes which may be controlled by the disclosed transcription factor active polypeptides including polypeptides such as, but not limited to, osteocalcin, xcex11 and xcex12, type I collagen, osteopontin, and bone sialoprotein.
In exemplary embodiments, the inventors have identified cell lines and cell types suitable for the present methods, including, but not limited to, Ros25, C3H10T42, C2C12, NIH3T3, F9, MC3T3E1, primary fibroblasts, myoblasts, chondrocytes, adipocytes, and marrow stromal cells.
In a third embodiment the invention provides methods for promoting the expression of an osteoblast-specific gene in a cell. These methods generally involve providing to a cell, an amount of an Osf2/Cbfa1 composition effective to promote the expression of the osteoblast-specific gene in the cell.
A fourth embodiment of the invention concerns a method for promoting the expression of a selected gene in a cell. The method generally involves providing to the cell, an expression system which contains one or more genes of interest which are positioned under the transcriptional control of an OSE2 element, and further providing to the cell an amount of an Osf2/Cbfa1 composition effective to promote the expression of the gene.
A fifth embodiment of the invention concerns a method of detecting a nucleic acid segment comprising at least one OSE2 element. This method generally involves contacting a population of nucleic acid segments suspected of containing one or more OSE2 elements with at least one Osf2/Cbfa1 composition under conditions and for a period of time effective to permit the binding of the Osf2/Cbfa1 composition(s) to the OSE2 element(s), and detecting the complex(es) so bound.
A sixth embodiment of the invention relates to a method of identifying an OSE2 element. This method generally involves contacting a sample suspected of containing an OSE2 element with an Osf2/Cbfa1 composition under conditions effective to allow binding of the Osf2/Cbfa1 composition to the OSE2 element, and detecting the bound complex.
The invention also provides a method of inducing osteoblast differentiation. This method comprises providing to an osteoblast progenitor cell an amount of an Osf2/Cbfa1 composition effective to induce differentiation of the progenitor cell. Exemplary cell cells include Ros25, C3H10T42, C2C12, NIH3T3, F9, MC3T3E1, primary fibroblast myoblasts, chondrocytes, adipocytes, and marrow stromal cells.
In yet another embodiment, there is provided a method for the production of an antibody that binds immunologically to an Osf2/Cbfa1 polypeptide, and in particular a mammalian Osf2/Cbfa1 polypeptide. This method generally comprises administering to an animal an immunologically-effective amount of an Osf2/Cbfa1 polypeptide composition. In one such method, co-administration of an adjuvant to the animal is contemplated to be particularly useful in producing an immune response in the animal, and the formation of antibodies specific for the particular Osf2/Cbfa1- or Osf2/Cbfa1-derived polypeptide, peptide, or epitope.
The invention also provides a method for identifying compounds which regulate, alter, or modulate the activity of an Osf2/Cbfa1 polypeptide or polynucleotide. This method generally comprises exposing a cell that expresses an Osf2/Cbfa1 polypeptide to at least one compound or signal whose ability to modulate the activity of the Osf2/Cbfa1 polypeptide is sought to be determined, and thereafter monitoring the cell for a change that is a result of the modulation of Osf2/Cbfa1 activity. Such an assay is particularly contemplated to be useful in the identification of agonists, antagonists and/or allosteric modulators of Osf2/Cbfa1. For example, recombinant Osf2/Cbfa1-producing cells may be contacted with one or more test compounds, and the modulating effect(s) thereof can then be evaluated by comparing the Osf2/Cbfa1-mediated response in the presence and absence of test compound, or relating the Osf2/Cbfa1-mediated response of test cells, or control cells (i.e. cells that do not express Osf2/Cbfa1), to the presence of the compound.
As used herein, a compound or signal that modulates the activity of Osf2/Cbfa1 refers to a compound that alters the activity of Osf2/Cbfa1 in such a way that the activity of Osf2/Cbfa1 is different in the presence of the compound or signal (as compared to the absence of said compound or signal).
A further aspect of the invention provides methods for screening compounds (e.g., synthetic peptides, peptide analogs, peptidomimetics, small molecule inhibitors, etc.) which inhibit or reduce the binding of an Osf2/Cbfa1 polypeptide with a polynucleotide. Being a promoter-specific transcription factor, Osf2/Cbfa1 is an important target for therapeutic intervention e.g., by means of a chemical entity affecting the factor""s capability of binding to the DNA. Therefore, the inventors contemplate that screening for such chemical entities may be performed e.g., by means of a cell-based assay, an in vitro assay for Osf2/Cbfa1 function and/or rational drug design. Cell-based assays for screening can be designed e.g., by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, is dependent on Osf2/Cbfa1. Such an assay enables the detection of compounds that directly antagonize Osf2/Cbfa1, or compounds that inhibit other cellular functions required for the activity of Osf2/Cbfa1.
In yet another embodiment, the present invention provides an isolated Osf2/Cbfa1 promoter element. Preferably the promoter element comprises a contiguous nucleic acid sequence of at least 17 nucleic acids from SEQ ID NO:72. As such, promoter elements contemplated to be useful in the practice of the present invention include those elements which comprise at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, and at least 26 contiguous nucleic acids from SEQ ID NO:72. Moreover, sequence elements which comprise at least 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or so contiguous nucleotides from SEQ ID NO:72 are also contemplated to be useful in the practice of the present invention, and are useful in promoting the expression of a gene operably linked to one or more of these promoter element sequences. This promoter element may comprise enhancer, inducer, and/or silencer elements that may be involved in controlling the cell-specific expression of the gene. In illustrative embodiments, the operably-linked gene may be any gene for which expression is desired to produce a polypeptide product from such gene. The gene may be a native, or mutated gene, and may be either homologous or heterologous. In certain embodiments, the expression of heterologous gene sequences from the Osf2/Cbfa1 promoter is contemplated to be useful in cells where expression of the gene under the control of its own promoter is either inefficient or impossible.
In illustrative embodiments, the Osf2/Cbfa1 promoter element may comprise a nucleic acid sequence having from about 60% to about 65%, to about 70%, to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, even up to and including about 96%, about 97%, about 98%, or about 99% or greater sequence identity with a contiguous nucleic acid sequence of at least about 17 or so nucleotides from SEQ ID NO:72. Of course, the percent identity to a contiguous nucleic acid sequence from SEQ ID NO:72 need not be limited to the specific percentages given, but is also meant to include all integers between about 60% and about 99% identity, such as percentage identities of about 86%, 87%, 88%, and 89%, or even about 91% or 92% or 93% or 94%, etc. identity with a contiguous nucleic acid sequence of at least about 17 or so nucleotides from SEQ ID NO:72. In fact, all such sequences are contemplated to fall within the scope of the present invention, so long as the particular sequence retains an ability to promote transcription of a nucleic acid segment operably linked to the DNA sequence comprising the Osf2/Cbfa1 promoter element. The inventors contemplate that the particular nucleic acid segment to be transcriptionally controlled (or promoted) by such an Osf2/Cbfa1 promoter polynucleotide may comprise one or more polynucleotides selected from the group consisting of homologous genes, heterologous genes, ribozymes, protein nucleic acids, and antisense constructs.
Another embodiment of the invention is an antisense nucleic acid segment which is complementary to a contiguous nucleotide sequence of at least about 17 or so nucleotides from SEQ ID NO:1 or SEQ ID NO:72. When it is desirable to negatively, or xe2x80x9cdown-regulatexe2x80x9d the expression of a particular gene or nucleic acid segment in a particular cell, the inventors contemplate that preparation of such antisense constructs will be useful in altering the activity of Osf2/Cbfa1 in a cell. Alternatively, if an antisense construct complementary to a contiguous nucleotide sequence from the Osf2/Cbfa1 promoter sequence (SEQ ID NO:72) such constructs may also be used to regulate the activity of any heterologous gene placed downstream of, and operably linked to, such an Osf2/Cbfa1 promoter.
Antisense constructs are well-known in the art, and in their simplest terms, relate to the use of antisense mRNA to reduce or lessen the transcription or translation or otherwise impair the net production of the encoded polypeptide. The preparation and use of such antisense constructs are described in detail hereinbelow.
A further embodiment of the invention concerns the preparation of ribozymes utilizing a promoter comprising a contiguous nucleotide sequence selected from SEQ ID NO:72. Means for preparing ribozymes using heterologous promoters operably linked to a ribozyme sequence are also well-known in the art, and described in detail hereinbelow.
In important aspects of the present invention, there are provided DNA constructs comprising one or more Osf2 promoters operably linked to or operatively positioned with respect to one or more heterologous genes. Exemplary heterologous genes which are contemplated to be useful include, but are not limited to, reporter genes such as GFP, GUS, lac, lux, xcex2-lactamase, xylE, xcex1-amylase, a tyrosinase gene, and aequorin; cell cycle control genes such as Rb, p53, a cell cycle dependent kinase, a CDK kinase or a cyclin gene.
A further aspect of the present invention provides a method of expressing a heterologous nucleic acid segment in a cell. The method generally involves transforming said cell with a vector comprising a heterologous nucleic acid segment operatively linked to at least one Osf2 promoter and culturing the cell under conditions effective to express the heterologous nucleic acid segment from the promoter. Preferably, the Osf2 promoter comprises a substantially contiguous nucleic acid sequence of at least about 17 contiguous nucleotides from SEQ ID NO:72 that retains the ability to promote transcription of a heterologous polynucleotide operably linked to the promoter. Most preferred are the smallest contiguous regions of SEQ ID NO:72 that retain the transcriptional activity of an Osf2/Cbfa1 promoter and that are capable of promoting the expression of such a heterologous gene. Preferably, the cell is an animal cell such as that from a human, monkey, hamster, caprine, feline, canine, equine, porcine, lupine, or murine. Of course, in certain embodiments, particularly in the preparation of recombinant vectors and the like, it may be desirable to prepare the constructs of the present invention for use in bacterial cells such as E. coli or salmonellas including S. typhimurium cells or in yeast.
In another embodiment the present invention provides a method of changing the characteristics of a cell. Characteristics include, but are not limited to, differentiation state, transformation state, color, fluorescence, antibiotic resistance, metabolic activity, or RNA expression profile.
In an illustrative embodiment, the present invention relates to a recombinant vector comprising an Osf2 promoter sequence from SEQ ID NO:72, or a substantially equivalent sequence that retains the transcriptional activity of the Osf2 promoter, operatively linked to a heterologous nucleic acid segment, in such an orientation as to control expression of said segment. The recombinant vector may be a plasmid, a cosmid, a YAC, a BAC, or a viral vector. Viral vectors include, but are not limited to, a bacteriophage vector, a Raus sarcoma virus vector, a p21 virus vector, an adeno-associated virus vector, and adenoviral vectors. Adenovirus vectors may be replication deficient of replication competent. In certain embodiments, the recombinant vector may be dispersed in a pharmaceutically acceptable solution.
The polynucleotides and proteins of the present invention may be used to identify molecules that control cell differentiation in the osteoblastic, chondrocytic and fibroblastic pathways. This can be achieved by ectopic expression (i.e. expression of the gene where it is normally not expressed) in cell culture studies and in transgenic mice. Moreover, the gene may also be used to screen (using a yeast two hybrid system, protein-protein interactions, or by immunoassay) for the proteins that interact with Osf2 or that regulate Osf2 expression or function. The nucleic acid compositions of the invention may also be used to identify regulatory sequences that control Osf2/Cbfa1 expression in cells of the osteoblastic lineage by DNA transfection studies and generation of transgenic mice lines. Antibodies generated against the novel protein may be used to perform DNA-binding assays to determine if the protein binds to other genes expressed in osteoblasts.
The invention provides nucleic acid sequences encoding an Osf2/Cbfa1 polypeptide. As used herein, an xe2x80x9cOsf2/Cbfa1 genexe2x80x9d means a nucleic acid sequence encoding an Osf2/Cbfa1 polypeptide. Preferred Osf2/Cbfa1 genes include mammalian Osf2/Cbfa1 genes, and in particular those from humans. A preferred nucleic acid sequence encoding an Osf2/Cbfa1 gene is the nucleotide sequence of SEQ ID NO:1 or substantially homologous variants, fusion proteins, or antigenically-active peptide fragments thereof. Also provided are nucleic acid sequences encoding an alternatively spliced variant of an Osf2/Cbfa1 polypeptide (SEQ ID NO:70) and a nucleic acid sequence that comprises an Osf2/Cbfa1 promoter (SEQ ID NO:72).
It is expected that the genes encoding Osf2/Cbfa1 polypeptides will vary in nucleic acid sequence from species to species, and even from strain to strain or cell line to cell line within a species, but that the variation in nucleic acid sequence will not preclude hybridization between sequences encoding the Osf2/Cbfa1 polypeptides of various species, cell lines, and strains under moderate to strict hybridization conditions. It is also contemplated that the genes encoding Osf2/Cbfa1 polypeptides from various strains may vary in nucleic acid sequences, but that the variation will not preclude hybridization between sequences encoding an Osf2/Cbfa1 polypeptides from various species, cell lines and strains under moderate to stringent hybridization conditions.
As used herein, a variant of an Osf2/Cbfa1 polypeptide means any polypeptide encoded, in whole or in part, by a nucleic acid sequence which hybridizes under moderate to stringent hybridization conditions to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:70, which encodes an Osf2/Cbfa1 polypeptide isolated from the human osteoblastic cell line designated SaOS, as well as from other human osteosarcoma cell lines.
One of skill in the art will understand that variants of Osf2/Cbfa1 polypeptides include those proteins encoded by nucleic acid sequences which may be amplified using one or more of the Osf2/Cbfa1 nucleic acid sequence disclosed in SEQ ID NO:1 or SEQ ID NO:70.
In related embodiments, the invention also comprises strain variants of Osf2/Cbfa1 polypeptides and nucleic acid segments encoding Osf2/Cbfa1 polypeptides, in particular, the Osf2/Cbfa1 genes which encode the Osf2/Cbfa1 polypeptide. The amino acid sequences of Osf2/Cbfa1 polypeptides claimed herein are disclosed in SEQ ID NO:2 (native Osf2/Cbfa1) and SEQ ID NO:71 (a splice variant of Osf2/Cbfa1).
Aspects of the invention concern the identification of such protein and peptide variants using diagnostic methods and kits described herein. In particular, methods utilizing Osf2/Cbfa1 gene sequences as nucleic acid hybridization probes and/or anti-Osf2/Cbfa1 antibodies in western blots or related analyses are useful for the identification of such variants. The identity of potential variants of Osf2/Cbfa1 polypeptides may also be confirmed by transcriptional assays as described in Section 5.
As used herein, an Osf2/Cbfa1 polypeptide means an isolated protein (or an epitope, variant, or active fragment thereof) derived from a mammalian species which has the ability to modulate osteoblast differentiation. Preferably, an Osf2/Cbfa1 polypeptide is encoded by a nucleic acid sequence having the sequence of SEQ ID NO:1 or SEQ ID NO:70, or a sequence which hybridizes to the sequence of SEQ ID NO:1 or SEQ ID NO:70. Alternatively, an Osf2/Cbfa1 polypeptide may be defined as a polypeptide which comprises a contiguous amino acid sequence from SEQ ID NO:2 or SEQ ID NO:71, or which protein comprises the entire amino acid sequence of SEQ ID NO:2 or SEQ ID NO:71.
In the present invention, an Osf2/Cbfa1 polypeptide composition is also understood to comprise one or more polypeptides that are immunologically reactive with antibodies generated against an Osf2/Cbfa1 polypeptide, particularly a protein having the amino acid sequence disclosed in SEQ ID NO:2 or SEQ ID NO:71; or the protein encoded by the Osf2/Cbfa1 nucleic acid sequence disclosed in SEQ ID NO:1 or SEQ ID NO:70, or to active fragments, or to variants thereof.
Likewise, an Osf2/Cbfa1 polypeptide composition of the present invention is understood to comprise one or more polypeptides that are capable of eliciting antibodies that are immunologically reactive with one or more Osf2/Cbfa1 polypeptides encoded by one or more contiguous Osf2/Cbfa1 nucleic acid sequences contained in SEQ ID NO:1 or SEQ ID NO:70, or to active fragments, or to strain variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency. Particularly preferred proteins include the amino acid sequence disclosed in SEQ ID NO:2 or SEQ ID NO:71.
As used herein, an active fragment of an Osf2/Cbfa1 polypeptide includes a whole or a portion of an Osf2/Cbfa1 polypeptide which is modified by conventional techniques, e.g., mutagenesis, or by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure and function as a native Osf2/Cbfa1 polypeptide as described herein.
Other aspects of the present invention concern isolated DNA segments and recombinant vectors encoding one or more Osf2/Cbfa1 polypeptides, in particular, the Osf2/Cbfa1 polypeptide from mammalian, and preferably, human sources, and the creation and use of recombinant host cells through the application of DNA technology, that express one or more Osf2/Cbfa1 gene products. As such, the invention concerns DNA segments comprising an isolated gene that encodes an Osf2/Cbfa1 polypeptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:2 or SEQ ID NO:71. These DNA segments are represented by those that include an Osf2/Cbfa1 nucleic acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:1 or SEQ ID NO:70, respectively.
Compositions that include a purified Osf2/Cbfa1 polypeptide that has a contiguous amino acid sequence essentially as set forth by the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:71 are also encompassed by the invention.
Regarding the novel Osf2/Cbfa1 polypeptides, the present invention concerns DNA segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode one or more proteins having osteoblast-specific transcription factor activity. DNA segments encoding one or more Osf2/Cbfa1-like species may also encode proteins, polypeptides, subunits, functional domains, antigenic epitopes, binding domains, and/or the like.
As used herein, the term xe2x80x9cDNA segmentxe2x80x9d refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding an Osf2/Cbfa1 polypeptide refers to a DNA segment that contains one or more Osf2/Cbfa1 coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term xe2x80x9cDNA segmentxe2x80x9d, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified Osf2/Cbfa1 gene refers to a DNA segment including Osf2/Cbfa1 coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. Preferably the sequence encodes an Osf2/Cbfa1 polypeptide, and more preferably, comprises an Osf2/Cbfa1 gene, in particular, an Osf2/Cbfa1 gene isolated from a mammalian cell line such as the Osf2/Cbfa1 gene isolated from human cell lines including one designated SaOS. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments may be naturally isolated, or modified synthetically by the hand of man.
xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case, a gene encoding an Osf2/Cbfa1 polypeptide, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode an Osf2/Cbfa1 polypeptide species that comprises an amino acid sequence essentially as set forth in SEQ ID NO:2 or SEQ ID NO:71, or biologically-functional equivalents thereof. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that comprises a sequence essentially as set forth in SEQ ID NO:1 or SEQ ID NO:70, or biologically-functional equivalents or strains variants thereof.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:1 or SEQ ID NO:70xe2x80x9d means that the sequence substantially corresponds to a portion of the DNA sequence listed in SEQ ID NO:1 or SEQ ID NO:70, and has relatively few nucleotides that are not identical to, or a biologically functional equivalent of, the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:70. Such nucleotide sequences are also considered to be essentially as those disclosed herein when they encode essentially the same amino acid sequences as disclosed, or that they encode biologically functional equivalent amino acids tot hose as disclosed herein. In particular, preferred nucleotide sequences are those which encode the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:71, or biologically functional equivalents thereof.
Likewise, the term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:70xe2x80x9d means that the sequence substantially corresponds to a portion of the DNA sequence listed in SEQ ID NO:70, and has relatively few nucleotides that are not identical to, or a biologically functional equivalent of, the nucleic acid sequence of SEQ ID NO:70. Such nucleotide sequences are also considered to be essentially as those disclosed herein when they encode essentially the same amino acid sequences as disclosed, or that they encode biologically functional equivalent amino acids tot hose as disclosed herein. In particular, preferred nucleotide sequences are those which encode the amino acid sequence of SEQ ID NO:71, or biologically functional equivalents thereof.
The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids disclosed herein, will be sequences that are xe2x80x9cessentially as set forth in SEQ ID NO:2xe2x80x9d or xe2x80x9cessentially as set forth in SEQ ID NO:71xe2x80x9d.
In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:1. The term xe2x80x9cessentially as set forth in SEQ ID NO:1xe2x80x9d is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1 and has relatively few nucleotides residues that are not identical, or functionally equivalent, to the nucleotide residues of SEQ ID NO:1. Again, DNA segments that encode proteins exhibiting an Osf2/Cbfa1 polypeptide-like activity will be most preferred.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various upstream or downstream regulatory or structural genes. It will also include a splice variant of an Osf2/Cbfa1 gene that has limited or no biologic activity, but which may act as a naturally-occurring xe2x80x9cdominant negativexe2x80x9d regulator of Osf2/Cbfa1 activity.
Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72. Nucleic acid sequences that are xe2x80x9ccomplementaryxe2x80x9d are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term xe2x80x9ccomplementary sequencesxe2x80x9d means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72, under relatively stringent conditions such as those described herein.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases. DNA segments with total lengths of about 2,000, about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
It will be readily understood that xe2x80x9cintermediate lengthsxe2x80x9d, in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001, 12,002, 13,001, 13,002 and the like.
It will also be understood that this invention is not limited to the particular nucleic acid sequence disclosed in SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72 or to the amino acid sequence disclosed in SEQ ID NO:2 or SEQ ID NO:71. Recombinant vectors and isolated DNA segments may therefore variously include the Osf2/Cbfa1 coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include an Osf2/Cbfa1 polypeptide coding region or may encode biologically functional equivalent polypeptides that have variant amino acids sequences.
The DNA segments of the present invention encompass biologically functional equivalent Osf2/Cbfa1 polypeptides and Osf2/Cbfa1-derived peptides, in particular those Osf2/Cbfa1 polypeptides isolated from mammals, and particularly humans. DNA segments isolated from mammalian species which are homologous to Osf2/Cbfa1-encoding nucleic acid sequences are particularly preferred for use in the methods disclosed herein. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent polypeptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
If desired, one may also prepare fusion proteins and peptides, e.g., where the Osf2/Cbfa1 coding regions are aligned within the same expression unit with other polypeptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter or an enhancer. The promoter (or enhancer) may be in the form of the promoter or enhancer that is naturally associated with an Osf2/Cbfa1 polypeptide gene (SEQ ID NO:72), as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein. The enhancer may be obtained by isolating the 5xe2x80x2 non-coding sequence located upstream of the coding sequence; by isolating the 3xe2x80x2 non-coding sequence located downstream of the coding sequences; or by isolating one or more intronic sequences located within the gene that contain one or more enhancer regions, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein.
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an Osf2/Cbfa1 gene in its natural environment. Such promoters may include Osf2/Cbfa1 promoters themselves, or promoters normally associated with other genes, and in particular other transcription factor genes, or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the Osf2/Cbfa1-encoding DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant polypeptides.
Prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promoter sequences such as those provided by tac, ara, trp, lac, lacUV5 or T7. When expression of the recombinant Osf2/Cbfa1 polypeptides is desired in eukaryotic cells, a number of expression systems are available and known to those of skill in the art. An exemplary eukaryotic promoter system contemplated for use in high-level expression is the Pichia expression vector system available from Pharmacial LKB Biotechnology.
In connection with expression embodiments to prepare one or more recombinant Osf2/Cbfa1 polypeptides or Osf2/Cbfa1-derived peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire Osf2/Cbfa1 polypeptide or one or more functional domains, epitopes, ligand binding domains, subunits, etc. therefore being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of an Osf2/Cbfa1 polypeptide or an Osf2/Cbfa1-derived peptide or epitopic core region, such as may be used to generate anti-Osf2/Cbfa1 antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 15 to about 100 amino acids in length, or more preferably, from about 15 to about 50 amino acids in length are contemplated to be particularly useful.
The Osf2/Cbfa1 gene and DNA segments derived therefrom may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the full length or active Osf2/Cbfa1 polypeptide is particularly contemplated. Expression of Osf2/Cbfa1 transgenes in animals is particularly contemplated to be useful in the production of anti-Osf2/Cbfa1 antibodies and the regulation or modulation of osteoblast differentiation.
In addition to their use in directing the expression of Osf2/Cbfa1, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to Osf2/Cbfa1-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow an Osf2/Cbfa1 polypeptide or regulatory gene product to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 14 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 14-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired.
Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72, or to any continuous portion of the sequence, from about 14-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the ternini of the total sequence.
The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO:1, SEQ ID NO:70, or SEQ ID NO:72, may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR(trademark) technology of U.S. Pat. No. 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire Osf2/Cbfa1 gene or gene fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50xc2x0 C. to about 70xc2x0 C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related Osf2/Cbfa1 genes.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate one or more Osf2/Cbfa1-encoding sequences from related species, functional equivalents, or the like, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20xc2x0 C. to about 55xc2x0 C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
Recombinant clones expressing the Osf2/Cbfa1-encoding nucleic acid segments may be used to prepare purified peptide antigens as well as mutant or variant protein species in significant quantities. The selected antigens, and variants thereof, are proposed to have significant utility in regulating, modulating, altering, changing, increasing, and/or decreasing osteoblast differentiation. For example, it is proposed that these antigens, or peptide variants, or antibodies against such antigens may be used in immunoassays to detect Osf2/Cbfa1 antibodies or as vaccines or immunotherapeutics to modulate osteoblast differentiation.
Additionally, by application of techniques such as DNA mutagenesis, the present invention allows the ready preparation of so-called xe2x80x9csecond generationxe2x80x9d molecules having modified or simplified protein structures. Second generation proteins will typically share one or more properties in common with the full-length antigen, such as a particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be provided on relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information. Such variant molecules may not only be derived from selected immunogenic/antigenic regions of the protein structure, but may additionally, or alternatively, include one or more functionally equivalent amino acids selected on the basis of similarities or even differences with respect to the natural sequence.
The Osf2 promoter may be used to express the Osf2/Cbfa1-encoding nucleic acid segments of the present invention. This allows the expression of these proteins to have the same tissue specificity and other activities as the endogenous gene. Similarly, one or more heterologous genes may be operably linked to the Osf2 promoter of the present invention to allow expression of one or more heterologous genes in a manner similar to that of the endogenous Osf2 gene.
Particular aspects of the invention concern the use of plasmid vectors for the cloning and expression of recombinant peptides, and particular peptides incorporating either native, or site-specifically mutated Osf2/Cbfa1 epitopes. The generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is well-known to those of skill in the art. Prokaryotic hosts are preferred for expression of the peptide compositions of the present invention. Some examples of prokaryotic hosts are E. coli strains JM101, XL1-Blue(trademark), RR1, LE392, B, "khgr"1776 (ATCC 31537), and W3110 (Fxe2x88x92, xcexxe2x88x92, prototrophic, ATCC 273325). Enterobacteriaceae species such as Salmonella typhimurium and Serratia marcescens, and other Gram-negative hosts such as various Pseudomonas species may also find utility in the recombinant expression of genetic constructs disclosed herein.
Alternatively, Gram-positive cocci such as S. aureus, S. pyogenes, S. dysgalactiae, S. epidermidis, S. zooepidemicus, S. xylosus, and S. hominus, and bacilli such as Bacillus subtilis, B. cereus, B. thuringiensis, and B. megaterium may also be used for the expression of these constructs and the isolation of native or recombinant peptides therefrom.
In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et al., 1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides ready means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as xcexGEM(trademark)-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
Those promoters most commonly used in recombinant DNA construction include the xcex2-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) or the tryptophan (trp) promoter system (Goeddel et al., 1980). The use of recombinant and native microbial promoters is well-known to those of skill in the art, and details concerning their nucleotide sequences and specific methodologies are in the public domain, enabling a skilled worker to construct particular recombinant vectors and expression systems for the purpose of producing compositions of the present invention.
In addition to the preferred embodiment expression in prokaryotes, eukaryotic microbes, such as yeast cultures may also be used in conjunction with the methods disclosed herein. Saccharomyces cerevisiae, or common baker""s yeast is the most commonly used among eukaryotic microorganisms, although a number of other species may also be employed for such eukaryotic expression systems. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschumper and Carbon, 1980). This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC 44076 or PEP4-1 (Jones, 1977). The presence of the trpL lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3N of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.
In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts in the routine practice of the disclosed methods. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
A particular aspect of this invention provides novel ways in which to utilize recombinant Osf2/Cbfa1-derived peptides, nucleic acid segments encoding these peptides, recombinant vectors and transformed host cells comprising Osf2/Cbfa1-derived DNA segments. As is well known to those of skill in the art, many such vectors and host cells are readily available, one particular detailed example of a suitable vector for expression in mammalian cells is that described in U.S. Pat. No. 5,168,050, incorporated herein by reference. However, there is no requirement that a highly purified vector be used, so long as the coding segment employed encodes a polypeptide of interest (e.g., an Osf2/Cbfa1-derived epitopic sequence) and does not include any coding or regulatory sequences that would have an adverse effect on cells. Therefore, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various regulatory sequences.
After identifying an appropriate epitope-encoding nucleic acid molecule, it may be inserted into any one of the many vectors currently known in the art, so that it will direct the expression and production of the polypeptide epitope of interest when incorporated into a host cell. In a recombinant expression vector, the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally associated with an Osf2/Cbfa1-encoding nucleic acid segment, as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein. Direct amplification of nucleic acids using the PCR(trademark) technology of U.S. Pat. Nos. 4,683,195 and 4,683,202 (each specifically incorporated herein by reference) are particularly contemplated to be useful in such methodologies.
In certain embodiments, it is contemplated that particular advantages will be gained by positioning the Osf2/Cbfa1-encoding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with an Osf2/Cbfa1 gene segment in its natural environment. Such promoters may include those normally associated with other transcription factor-encoding genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising an Osf2/Cbfa1 epitope-encoding nucleic acid segment.
The use of recombinant promoters to achieve protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment. For eukaryotic expression, preferred promoters include those such as a CMV promoter, an RSV LTR promoter, a xcex2-actin promoter, an insulin promoter, an SV40 promoter alone, or an SV40 promoter either alone, or in combination with one or more enhancers, such as an SV40 enhancer. Prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promoter sequences such as a tac, ara, trp, lac, lacUV5 or T7 promoter.
Another aspect of the present invention includes novel compositions comprising isolated and purified Osf2/Cbfa1 polypeptides, Osf2/Cbfa1-derived peptides, synthetic modifications of these epitopic peptides, peptides derived from site-specifically-mutagenized nucleic acid segments encoding such peptides, polynucleotides, and/or antibodies specific for Osf2/Cbfa1 and Osf2/Cbfa1-derived peptides and polypeptides. It will, of course, be understood that one or more than one Osf2/Cbfa1-encoding nucleic acid segment may be used in the methods and compositions of the invention. The nucleic acid delivery methods may thus entail the administration of one, two, three, or more, Osf2/Cbfa1 nucleic acid segments encoding one or more transcription factors. The maximum number of nucleic acid segments that may be applied is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of nucleic acid segment constructs or even the possibility of eliciting an adverse cytotoxic effect.
The particular combination of nucleic acid segments may be two or more distinct nucleic acid segments; or it may be such that a nucleic acid segment from one gene encoding Osf2/Cbfa1 is combined with another nucleic acid segment and/or another peptide or protein such as a cytoskeletal protein, cofactor targeting protein, chaperone, or other biomolecule such as a vitamin, hormone or growth factor gene. Such a composition may even further comprise one or more nucleic acid segments or genes encoding portions or all of one or more cell-surface receptors or bone-specific targeting proteins capable of interacting with the polypeptide product of the Osf2/Cbfa1-encoding nucleic acid segment.
In using multiple nucleic acid segments, they may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same or different types. Thus, an almost endless combination of different nucleic acid segments and genetic constructs may be employed. Certain combinations of nucleic acid segments may be designed to, or their use may otherwise result in, achieving synergistic effects on inhibiting osteoblast differentiation and/or stimulation of an immune response against peptides derived from translation of such nucleic acid segments. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic combinations of nucleic acid segments, or even nucleic acid segment-peptide combinations.
It will also be understood that, if desired, the nucleic acid segment or gene encoding a particular Osf2/Cbfa1-derived peptide may be administered in combination with further agents, such as, e.g., proteins or polypeptides or various pharmaceutically-activeagents. So long as the composition comprises a nucleic acid segment encoding all or portions of an Osf2/Cbfa1 polypeptide, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The nucleic acids may thus be delivered along with various other agents as required in the particular instance. The formulation of pharmaceutically-acceptable excipients and carrier solutions are well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions herein described in oral, parenteral, and/or intravenous administration and formulation.
The Osf2/Cbfa1 pharmaceutical compositions disclosed herein may be prepared and delivered in a variety of formulations and methods depending upon the particular application. For example, in the case of oral administration, the disclosed compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration,the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
Likewise, for oral administration, the compositions of the invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell""s Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate, dispersed in dentifrices, including: gels, pastes, powders and slurries, or added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
Alternatively, in certain embodiments, it may be desirable to administer the Osf2/Cbfa1 pharmaceutical compositions disclosed herein either parenterally, intravenously, intramuscularly, or even intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
As used herein, xe2x80x9ccarrierxe2x80x9d includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifingal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The phrase xe2x80x9cpharmaceutically-acceptablexe2x80x9d refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.
The invention also encompasses Osf2/Cbfa1-derived peptide antigen compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, such as additional peptides, antigens, cell membrane preparations, or even attenuated whole-cell compositions as may be employed in the formulation of particular vaccines.
Using the peptide antigens described herein, the present invention also provides methods of generating an immune response, which methods generally comprise administering to an animal, a pharmaceutically-acceptable composition comprising an immunologically effective amount of an Osf2/Cbfa1-derived peptide composition. Preferred animals include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, porcines, canines, and felines. The composition may include partially or significantly purified Osf2/Cbfa1-derived peptide epitopes, obtained from natural or recombinant sources, which polypeptides may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes. Smaller peptides that include reactive epitopes, such as those between about 30 and about 100 amino acids in length will often be preferred. The antigenic polypeptides may also be combined with other agents, such as other peptides or nucleic acid compositions, if desired.
By xe2x80x9cimmunologically effective amountxe2x80x9d is meant an amount of a peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various therapeutic embodiments.
Further means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a peptide epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition. The xe2x80x9cimmunologically effective amountsxe2x80x9d are those amounts capable of stimulating a B cell and/or T cell response.
Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies specific to Osf2/Cbfa1 and related proteins. Antigenic functional equivalents of these proteins and peptides also fall within the scope of the present invention. An xe2x80x9cantigenically functional equivalentxe2x80x9d polypeptide is one that incorporates an epitope that is immunologically cross-reactive with one or more epitopes derived from the Osf2/Cbfa1 polypeptides disclosed. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity.
The identification or design of suitable Osf2/Cbfa1 epitopes, and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of Hopp (as disclosed in U.S. Pat. No. 4,554,101, which is specifically incorporated herein by reference) in the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. These methods, described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences. For example, Chou and Fasman (1974a,b; 1978a,b; 1979); Jameson and Wolf (1988); Wolf et al. (1988); and Kyte and Doolittle (1982) all address this subject in several scientific publications. The amino acid sequence of these xe2x80x9cepitopic core sequencesxe2x80x9d may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
It is proposed that the use of shorter antigenic peptides, e.g., about 25 to about 50, or even about 15 to 25 amino acids in length, that incorporate modified epitopes of Osf2/Cbfa1 will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is contemplated that the polypeptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect Osf2/Cbfa1 polypeptides. Either type of kit may be used in the immunodetection of Osf2/Cbfa1 compositions. The kits may also be used in antigen or antibody purification, as appropriate.
In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing an Osf2/Cbfa1-specific antibody, such as a biological sample from a patient, and contacting the sample with a first Osf2/Cbfa1 polypeptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed.
Contacting the chosen sample with the Osf2/Cbfa1-derived polypeptide under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the polypeptide composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens to form immune complexes with, i.e. to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antigen species, allowing only those specifically bound species within the immune complexes to be detected.
The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as, e.g., Nakamura et al. (1987), incorporated herein by reference. Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.
Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first polypeptide. The second binding ligand is itself often an antibody, which may thus be termed a xe2x80x9csecondaryxe2x80x9d antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.
For diagnostic purposes, it is proposed that virtually any sample suspected of containing either the antibodies of interest may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. This allows for the diagnosis of meningitis, otitis media, pneumonia, bacteremia and postpartum sepsis. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats. Samples from feline, canine, and equine sources may also be used in accordance with the methods described herein.
In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of Osf2/Cbfa1-derived epitope-specific antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable polypeptide together with an immunodetection reagent, and a means for containing the polypeptide and reagent.
The immunodetection reagent will typically comprise a label associated with an Osf2/Cbfa1 polypeptide, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first Osf2/Cbfa1 polypeptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g., for protocols where the first reagent is an Osf2/Cbfa1 peptide that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
In another aspect, the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention. As stated above, one of the uses for Osf2/Cbfa1 peptides according to the present invention is to generate antibodies. Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies, and parts thereof, either alone or conjugated with other moieties. Antibody parts include Fab and F(ab)2 fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a polyclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, 1988).
Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
Antibodies, both polyclonal and monoclonal, specific for Osf2/Cbfa1 and Osf2/Cbfa1-derived peptides and/or epitopes may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic Osf2/Cbfa1 epitopes can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against epitope-containing Osf2/Cbfa1 peptides. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (below).
One of the important features provided by the present invention is a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein. Typically, polyclonal antisera is derived from a variety of different xe2x80x9cclones,xe2x80x9d i.e. B-cells of different lineage. Monoclonal antibodies, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their xe2x80x9cmonoxe2x80x9d clonality.
When peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non-native) conformations. As a result, even short peptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule.
Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention provides for polyclonal antisera where the clonality, i.e. the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonalityxe2x80x9490%, 95% or greaterxe2x80x94is contemplated.
To obtain monoclonal antibodies, one would also initially immunize an experimental animal, often preferably a mouse, with an Osf2/Cbfa1 polypeptide or Osf2/Cbfa1-derived peptide or epitope-containing composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.
Following immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against Osf2/Cbfa1-derivedepitopes. Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as immunoprecipitation, ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the Osf2/Cbfa1 and Osf2/Cbfa1-derived epitope-specific monoclonal antibodies.
It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as immunoprecipitation, ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to the Osf2/Cbfa1 or Osf2/Cbfa1-derived epitopes.
Additionally, it is proposed that monoclonal antibodies specific to the particular Osf2/Cbfa1-derived peptide may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant peptide species or synthetic or natural variants thereof.
In general, both poly- and monoclonal antibodies against these peptides may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding the peptides disclosed herein or related proteins. They may also be used in inhibition studies to analyze the effects of Osf2/Cbfa1-derived peptides in cells or animals. Anti-Osf2/Cbfa1 epitope antibodies will also be useful in immunolocalization studies to analyze the distribution of Osf2/Cbfa1 various cellular events, for example, to determine the cellular or tissue-specific distribution of the Osf2/Cbfa1 peptides under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant Osf2/Cbfa1 or Osf2/Cbfa1-derived peptides, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund""s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund""s adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately about 5xc3x97107 to about 2xc3x97108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4-1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (vol./vol.) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).
Fusion procedures usually produce viable hybrids at low frequencies, about 1xc3x9710xe2x88x926 to about 1xc3x9710xe2x88x928. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two wk. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three wk) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines may also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
The present invention is also directed to Osf2/Cbfa1 polypeptide compositions, free from total cells and other polypeptides, which comprise a purified Osf2/Cbfa1 polypeptide which incorporates an epitope that is immunologically cross-reactive with one or more of the Osf2/Cbfa1-specific antibodies of the present invention.
As used herein, the term xe2x80x9cincorporating an epitope(s) that is immunologically cross-reactive with one or more anti-Osf2/Cbfa1 antibodiesxe2x80x9d is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within an Osf2/Cbfa1 polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the Osf2/Cbfa1 polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
The identification of Osf2/Cbfa1 epitopes and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these xe2x80x9cepitopic core sequencesxe2x80x9d may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a xe2x80x9cuniversalxe2x80x9d epitopic peptide directed to Osf2/Cbfa1-related sequences. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation in an animal, and, hence, elicit specific antibody production in such an animal.
An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is xe2x80x9ccomplementaryxe2x80x9d to, and therefore will bind, antigen binding sites on Osf2/Cbfa1 epitope-specific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term xe2x80x9ccomplementaryxe2x80x9d refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence expected by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysis programs (e.g., DNAStar(trademark) software, DNAStar, Inc., Madison, Wis.) may also be useful in designing synthetic epitopes and epitope analogs in accordance with the present disclosure.
The peptides provided by this invention are ideal targets for use as vaccines or immunoreagents for the regulation or modulation of osteoblast differentiation, and in particular, that caused by the transcription factor Osf2/Cbfa1. In this regard, particular advantages may be realized through the preparation of synthetic Osf2/Cbfa1 peptides that include epitopic/immunogenic core sequences. These epitopic core sequences may be identified as hydrophilic and/or mobile regions of the polypeptides or those that include a T cell motif. It is known in the art that such regions represent those that are most likely to promote B cell or T cell stimulation, and, hence, elicit specific antibody production.
To confirm that a polypeptide is immunologically cross-reactive with, or a biological functional equivalent of, one or more epitopes of the disclosed peptides is also a straightforward matter. This can be readily determined using specific assays, e.g., of a single proposed epitopic sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments. The screening assays may be employed to identify either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, i.e. based upon competition for binding sites between antibodies and antigens.
Suitable competition assays that may be employed include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like. In any of the competitive assays, one of the binding components, generally the known element, such as an Osf2/Cbfa1 or Osf2/Cbfa1-derived peptide, or a known antibody, will be labeled with a detectable label and the test components, that generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen.
As an exemplary embodiment, to conduct a competition study between Osf2/Cbfa1 and any test antigen, one would first label Osf2/Cbfa1 with a detectable label, such as, e.g., biotin or an enzymatic, radioactive or fluorogenic label, to enable subsequent identification. One would then incubate the labeled antigen with the other, test, antigen to be examined at various ratios (e.g., 1:1, 1:10 and 1:100) and, after mixing, one would then add the mixture to a known antibody. Preferably, the known antibody would be immobilized, e.g., by attaching to an ELISA plate. The ability of the mixture to bind to the antibody would be determined by detecting the presence of the specifically bound label. This value would then be compared to a control value in which no potentially competing (test) antigen was included in the incubation.
The assay may be any one of a range of immunological assays based upon hybridization, and the reactive antigens would be detected by means of detecting their label, e.g., using streptavidin in the case of biotinylated antigens or by using a chromogenic substrate in connection with an enzymatic label or by simply detecting a radioactive or fluorescent label.
The reactivity of the labeled antigen, e.g., an Osf2/Cbfa1-derived peptide, in the absence of any test antigen would be the control high value. The control low value would be obtained by incubating the labeled antigen with an excess of unlabeled antigen, when competition would occur and reduce binding. A significant reduction in labeled antigen reactivity in the presence of a test antigen is indicative of a test antigen that is xe2x80x9ccross-reactivexe2x80x9d, i.e. that has binding affinity for the same antibody. xe2x80x9cA significant reductionxe2x80x9d, in terms of the present application, may be defined as a reproducible (i.e. consistently observed) reduction in binding.
In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially-available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4xc2x0 C., or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.
As mentioned, in certain aspects, the DNA sequence information provided by the present disclosure allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to nucleic acid sequences encoding portions of the Osf2/Cbfa1 gene, which the inventors have identified as an osteoblast-specific transcription factor. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of the natural sequence and the size of the particular DNA segment used. Such DNA segments may be those of native Osf2/Cbfa1 or Osf2/Cbfa1-derived, or alternatively, may be DNA sequences which have undergone site-specific mutations to generate any of the novel peptides disclosed herein. The ability of such nucleic acid probes to specifically hybridize to the corresponding Osf2/Cbfa1 nucleic acid sequences lend them particular utility in a variety of embodiments. However, other uses are envisioned, including the expression of protein products, the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions. Such primers may also be used as diagnostic compositions for the isolation and identification of epitope-encoding nucleic acid segments from transcription factors related to Osf2/Cbfa1.
To provide certain of the advantages in accordance with the present invention, the preferred nucleic acid sequence employed for hybridization studies or assays would include sequences that have, or are complementary to, at least an about 14 or 15 to about 20 or so nucleotide stretch of the sequence, although sequences of about 30 to about 50 or so nucleotides are also envisioned to be useful. A size of at least 14-15 or 20 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 14-15 or 20 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. Thus, one will generally prefer to design nucleic acid molecules having Osf2/Cbfa1-gene-complementary stretches of 14-15 to 20-25 nucleotides, or even longer, such as about 30, or about 50, or about 100, or even about 200 nucleotides, where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR(trademark) technology of U.S. Pat. No. 4,683,202, or by introducing selected sequences into recombinant vectors for recombinant production.
The inventors further contemplate that such DNA segments will have utility in the overexpression of Osf2/Cbfa1-derived peptide epitopes described herein, and the preparation of recombinant vectors containing native and site-specific-mutagenized DNA segments comprising particular epitope regions from the Osf2/Cbfa1 gene.
The invention will find particular utility as the basis for diagnostic hybridization assays for detecting Osf2/Cbfa1-specific RNA or DNA in clinical samples. Exemplary clinical samples that can be assayed for the presence of Osf2/Cbfa1 or Osf2/Cbfa1-encoding nucleic acids include middle ear fluid, sputum, bronchoalveolar fluid and the like. Such samples may be of human, murine, equine, bovine, feline, porcine, or canine origins. A variety of hybridization techniques and systems are known that can be used in connection with the hybridization aspects of the invention, including diagnostic assays such as those described in U.S. Pat. No. 4,358,535, incorporated herein by reference. Samples derived from non-human mammalian sources, including animals of economic significance such as domestic farm animals, may also provide the basis for clinical specimens.
Accordingly, the nucleotide sequences of the invention are important for their ability to selectively form duplex molecules with complementary stretches of the nucleic acid segments encoding Osf2/Cbfa1 epitopes. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent hybridization conditions are called for in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ lower or reduced stringency hybridization conditions. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, one may desire to employ nucleic acid probes to isolate variants from clone banks containing mutated Osf2/Cbfa1-encoding clones. In particular embodiments, mutant clone colonies growing on solid media that contain variants of the Osf2/Cbfa1 gene could be identified on duplicate filters using hybridization conditions and methods, such as those used in colony blot assays, to only obtain hybridization between probes containing sequence variants and nucleic acid sequence variants contained in specific colonies. In this manner, small hybridization probes containing short variant sequences of these genes may be utilized to identify those clones growing on solid media that contain sequence variants of the entire genes. These clones can then be grown to obtain desired quantities of the variant nucleic acid sequences or the corresponding antigens.
In clinical diagnostic embodiments, nucleic acid sequences of the present invention are used in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, that are capable of giving a detectable signal. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with pathogen nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) from suspected clinical samples, such as exudates, body fluids (e.g., middle ear effusion, bronchoalveolarlavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.